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Application No. 2

Sample Preparation for Microinjection

General Comments Microinjection is the loading or transfer of a dissolved substance into a living cell. The microscopic tip of the glass microcapillary has an inner diameter between 0.2 and 1 µm [1]. This capillary is back loaded with the substance to be transferred into the cells cultured for microinjection. Typical substances include purified antibodies, DNA, RNA, peptides, or oligonucleotides.

To visualize and evaluate the success of a microinjection experiment these substances are typically mixed with dyes or labeled with fluorescent markers such as flourescein or rhodamine. After the capillary has pierced the cell, a certain amount of transfer substance (approximately 10% of the cell volume) is transferred from the capillary into the cell due to pressure exerted on the capillary via the microinjector [2]. Because the diameter of the capillary is very small, particles in the injection solution can quickly result in blockage of the capillary. Therefore, exchange of the capillary is a common practice during microinjection experiments and the frequency of the capillary exchange depends on the concentration and clarity of substance injected.

To maximize the number of injections which can be performed with a single capillary, the injection substance should be centrifuged for 1015 minutes at a minimum of 10,000 x g before back loading the capillary. As the cross section of the capillary tip is small and very little liquid would be found there, it dries quickly and creates a blockage. Therefore, the filled capillary should be inserted into the capillary holder and immediately lowered into the medium to avoid blockage due to drying. The capillary should always remain in the medium especially during long experiments when large numbers of cells are injected.

In order that the open petri dish has a sufficient pH stability under the microscope, a carbonate-free medium must be used (e.g., HEPES, pH 7.4 buffered, 10 mmol/l). A 3 cm dish should be filled with at least 2 ml medium. Cultivation can still take place in the usual medium containing carbonate.

The speed and efficiency of automated systems allows biochemical analysis of microinjected cells. Between 500 and 1000 labeled cells are sufficient to analyze labeled proteins by gel electrophoresis.

Place coverslips into a humid chamber and incubate at 37°C until cells attach to the glass (usually takes 68 hr).

Transfer coverslips into 35 mm petri dishes containing 2 ml of culture medium and let cells grow for 2 days at 37°C. After this time, 500 to 1000 cells will usually be in the center of the coverslip.

Microinject all cells on the coverslip.

Proceed with biochemical analysis (depends on the particular experiment).

Note: For time series experiments, the cells may be plated directly on to Eppendorf® CELLocate gridded cover slips placed in the center of a glass slide. Thereby individual cells (55 µm) or cell groups (175 µm) can be located easily after microinjection. Eppendorf Micromanipulator 5179 and FemtoJet can be used with Femtotips and Femtotips II precision microcapillaries to efficiently perform cytoplasmic and nuclear microinjection on a wide range of adherent cells.

Proteins, Antibodies Purification [4] Purification methods which result in the highest activity of the particular proteins or antibodies should be used. For peptide antibodies raised in rabbits, affinity purification is recommended. The concentration of the material injected should be 10 to 20 times higher than that required for the optimum in vitro activity, because the sample is diluted 10 to 20 times when injected into the cells.

Storage Purified antibodies must be stored in the concen-tration in which they arise. Shock freeze small aliquots of 510 µl in liquid nitrogen. Store at maximum temp. of 20°C (80°C is even better). Azide should not be used.

Refrain from repeated freezing and defrosting as antibodies lose activity and start to agglutinate leading to blockages of the injection capillaries.

DNA [5] Purification The highest expression level of microinjected plasmid DNA is achieved when the DNA has been purified by CsCl ethidium bromide gradient centrifugation in accordance with the Maniatis protocol [6].

Dissolve DNA in bidist. water after purification.

Buffers, e.g., PBs, are not recommended as this often leads to blockages of the capillaries.

DNA concentrations of 20 µg/ml to 200 µg/ml for plasmid DNA injection has been recommended [1].

Dextran marked e.g. with rhodamine of a concentration of 2 µg/ml can be detected for up to 48 hours in the cell.

Fluorescein fades with time and results in harmful radicals, therefore markers should preferably be labeled with rhodamine.

Storage Store at 80°C in small aliquots of 510 µl.

Many solutions are sensitive to light. Thus, exposure to direct light should be avoided.

To avoid blocking of the capillary during microinjection, solutions containing the markers should be filtered with a syringe filter (pore size 0.2 µm) whenever possible. Also, before loading the capillary, the sample should be centrifuged for 15 minutes at 4°C (10,000 x g).

Peptides Please refer to the current literature for information on the cleaning and use of peptides.

A concentration of at least 510 µg/ml should be used for injection, because some peptides rapidly degrade in the cells after injection.

Oligonucleotides Purification The purification of oligonucleotide solutions is very important. Cleaning with gel or HPLC is recommended.

Like DNA, oligonucleotides are dissolved in bidist. water after purification.

A concentration of 12 µg/ml should be used for injection of antisense oligonucleotides with 1020 bases.